Ambion’s innovative Cells-to-cDNA technology provides a simple and rapid method for preparing cultured cell lysates that can be used directly in gene expression analysis studies. Paired with Applied Biosystems reagents for reverse transcription, TaqMan-based real-time PCR, and real-time PCR instruments, you can generate reliable, quantitative, gene expression data more rapidly than ever before. We compared expression data for 24 genes in seven cell lines using either DNase-treated cell lysates, generated with our Cells-to-cDNA II Cell Lysis Buffer, or purified RNA isolated using a kit from Supplier Q. Samples derived from a broad range of cell concentrations were reverse transcribed using Applied Biosystems High Capacity cDNA Reverse Transcription Kit. cDNA was then amplified using TaqMan® Universal PCR Master Mix and a panel of TaqMan Gene Expression Assays (both from Applied Biosystems). Expression level, linear response to sample dilution, and variance among technical replicates were comparable between the two methods. Using this approach, we were able to corroborate published observations suggesting that EGFR is highly expressed in HeLa cells and not expressed, or expressed at very low levels, in Raji cells.

The Cuff laboratory combined two isolation techniques to obtain pure populations of DCs. An anti-CD11c MACS column (Miltenyi Biotech) was used to selectively bind DCs present in dissected Peyers's Patches from infected or uninfected animals. The DCs were eluted from the column and further enriched using fluorescent activated cell sorting (FACS) based on differential expression of characteristic myeloid and lymphoid markers. The purification strategy resulted in less than 10,000 cells per sample. To avoid the inevitable loss of RNA using standard RNA isolation protocols, the samples were processed using Cells-to-cDNA technology. In brief, each cell sample was lysed in 50 µl of Cells-to-cDNA Lysis Buffer by heating sample at 75°C for 10 min. DNase I digestion was performed at 37°C for 30 min. Reverse transcription of 10 µl of the lysate was followed by real-time PCR using commercial TaqMan® probes (ABI) and primers in a Roche Lightcycler®.

Gene Expression Analysis Without RNA Isolation

Traditionally, the first step in gene expression experiments is isolation of RNA from samples. Even using the quickest and simplest techniques, RNA isolation requires significant hands-on manipulation and time. Ambion, an Applied Biosystems Business, has developed an innovative reagent, Cells-to-cDNA II Cell Lysis Buffer, that lyses cultured mammalian cells while protecting endogenous RNA from degradation. The lysate serves as an effective template for real-time RT-PCR without the need for RNA purification. Cells-to-cDNA II lysates can be efficiently reverse transcribed using the robust High Capacity cDNA Reverse Transcription Kit. Then, quantitative gene expression levels can be determined using TaqMan® Universal PCR Master Mix, state-of-the-art TaqMan Gene Expression Assays, and trusted real-time PCR instruments. Furthermore, this protocol can be automated for high throughput screening applications. Here, we present a series of experiments that test the RNA isolation-free method for gene expression analysis (Figure 1) in several cultured mammalian cell lines across a wide range of cell inputs with up to 24 TaqMan Gene Expression Assays.

Figure 2 outlines the process for comparing gene expression data from RNA purified with the Supplier Q Kit versus direct real-time RT-PCR using cell lysates prepared with Ambion Cells-to-cDNA II Cell Lysis Buffer and DNase I. Cells from the 7 cell lines shown in Figure 3 were collected, diluted with culture media to final cell concentrations of 10-300,000 cells per lysis, washed with PBS, and lysed in Cells-to-cDNA II Cell Lysis Buffer. Lysates were then treated with DNase I. By diluting cells before lysis, the reagent concentrations in the lysate samples were kept equivalent, and cell number effects on lysis were captured. To obtain purified RNA, cells were processed using the Supplier Q Kit according to the manufacturer’s instructions. The purified RNA was then diluted in water to mimic the cell concentrations used in the cell lysate method.

Cell Line

Type

Source

HeLa

adherent

Cervical Adenocarcinoma

Raji

suspension

Burkitt's Lymphoma

Jurkat

suspension

T-Cell Leukemia

MCF-7

adherent

Breast Adenocarcinoma

HEPG2

adherent

Liver Carcinoma

293

adherent

Embryonic Kidney

U87-MG

adherent

Neuronal

Figure 3. Cell Lines Used in the Study.

Step 1: Cell Lysis. The Cells-to-cDNA II cell lysate method requires only three pipetting and/or heating steps. Isolating RNA using the Supplier Q Kit requires at least 8 steps, not including removing wash solutions from each collection tube after centrifugation. Sample prep using the cell lysate method is significantly faster and requires much less manual manipulation than RNA purification. Less sample handling and hands-on time is a big advantage, reducing variability during sample preparation and decreasing chances of sample mix-up.

Step 2: Reverse Transcription. After sample preparation was complete, samples (10 µL aliquots) were reverse transcribed in 50 µL reactions using the High Capacity cDNA Reverse Transcription Kit. The kit’s capacity, efficiency, and rigorous linearity enable it to synthesize maximal yields of cDNA from the wide range of sample amounts used in this study.

Step 3: Detection Using Real-time PCR. Finally, 10 µL of each reverse transcription reaction served as template in a real-time PCR procedure using one of 16 TaqMan Gene Expression Assays (Figure 4). Each 100 µL lysate or RNA sample provided template to assay gene expression levels from 8 genes. There were 4 technical replicates of each TaqMan Assay; each PCR amplification contained 2% of the initial 100 µL sample. The reactions were run with TaqMan Universal PCR Master Mix on a 7900HT Fast Real-Time PCR System under universal cycling conditions.

Results. Figure 5 shows a representative subset of the gene expression data obtained with Cells-to-cDNA II lysates or purified RNA isolated from 7 cell lines and tested at 10 dilutions using 16 TaqMan Gene Expression Assays with 4 technical replicates. Both sample types show extremely similar trend lines. Although the Ct values were often 1-2 Ct higher for the lysate samples, the slope and linearity of the assay results were comparable. Furthermore, we obtained good sensitivity and reproducibility across the entire range of cell amounts tested in these experiments: 10-300,000 cells per 100 µL lysis reaction.

Figure 5. Cell Lysates without RNA Isolation Give Comparable Gene Expression Data to Purified Total RNA. Quantitative gene expression data for 12 TaqMan® Gene Expression Assays conducted on 10 increasing quantities of HeLa cells. Plots of Ct vs. cell number for the indicated TaqMan Assay compare data from the Cells-to-cDNA™ II cell lysate method to that obtained from purified RNA with the same RT-PCR reagents. Similar results were obtained for six other cell lines (data not shown).

Conclusions. Our analysis determined the following:

The slopes of Ct vs. cell number for lysates are comparable to those for purified RNA. This indicates that the complex mixture of biomolecules in Cells-to cDNA II cell lysate samples does not affect RT-PCR efficiency.

Analysis of the correlation between cell number and Ct showed that >95% of our assays have a linear dynamic range that extends to input levels of 30,000 cells per lysis. This result confirmed that residual cellular debris had no detectable influence on RT-PCR linearity up to this concentration.

Even at 300,000 cells per lysis, which roughly corresponds to the number of cells in 12 confluent wells of a 96-well plate, >50% of the TaqMan Assays and cell line combinations tested maintained full linearity. (It is important to evaluate a range of cell numbers to determine the dynamic range of your own system.) Figure 6 shows the correlation coefficient (R2) of the TaqMan Assays across all cell lines for cell lysates and purified RNA.

Variance among technical replicates for cell lysates and purified RNA was the same.

Figure 6. Linearity is Equivalent For Cells-to-cDNA™ II Lysates and Purified Total RNA. R2 describes the correlation between the measured Ct values and the log of the reaction template quantity. It is a statistical estimate of the proximity of the Ct measurements to linearity across the number of cells used. This graph shows the density of R2 values compiled from all study experiments. The pattern from both Cells-to-cDNA II lysates and purified RNA is identical and indicates that the gene expression data from lysates maintains linearity.

Even for Low-Expressing Genes, Conclusions Were Equivalent for Cell Lysates and Purified RNA

Next we tested whether the RNA isolation-free method (Cells-to-cDNA protocol) would also work well for expression studies of genes characterized as low expressers. Using the same experimental design and dilution series as in the initial set of experiments (Figure 2), we assayed HeLa and Raji cells for expression of eight genes known to be expressed at relatively low levels (Figure 7). As in the initial study, the results from Taqman Gene Expression Assays conducted using cDNA generated from Cells-to-cDNA II lysates or purified RNA were in close agreement (Figure 8). Overall, our analysis determined that the Ct values, standard deviation among technical replicates, and slopes and linearity of low expresser assay results were no different for the two sample preparation methods. These results indicate that, despite their relatively low concentration in the complex mix of cellular debris in the cell lysates, there is no differential loss of signal for low expressing genes in comparison to purified RNA. Thus the RNA isolation-free method can be used with these more challenging targets as well as for higher expressing genes.

Figure 8. Gene Expression Data from Low Expressing Genes Is Equivalent Using Cells-to-cDNA™ II Lysates or Purified Total RNA. Quantitative gene expression data for 6 TaqMan® Gene Expression Assays conducted on 10 increasing quantities of HeLa cells. Plots of Ct vs cell number for the indicated TaqMan Assay compare data from an RT-PCR experiment using the Cells-to-cDNA II cell lysates or purified RNA. Similar results were obtained in six other cell lines (data not shown).

A Simpler, Faster Way to Do Gene Expression Analysis

In this study we were interested in whether samples prepared in Ambion Cells-to-cDNA II Cell Lysis Buffer would perform well with Applied Biosystems reverse transcription and TaqMan Gene Expression Assay reagents. The results were clear. Not only did the Cells-to-cDNA II lysate samples perform well, gene expression data using the RNA isolation-free procedure in which cell lysates are used directly in real-time RT-PCR were comparable to data obtained using total RNA purified with the Supplier Q Kit. These results indicate that RNA isolation from cultured mammalian cells is not necessary for gene expression analysis using TaqMan Assays. Using this combination of Ambion and Applied Biosystems products, we were able to screen several cell lines quickly across a number of TaqMan Assays. Results were comparable to those using purified RNA, and good linearity was observed across a cell concentration gradient of 10-300,000 cells per 100 µL lysis. The Cells-to-cDNA II lysis protocol dramatically reduced the steps required for sample preparation, thus reducing handling time. The Cells-to-cDNA II lysate was similar to purified RNA in reverse transcription efficiency and showed little or no PCR inhibition even at high cell concentrations. When cDNA generated from the lysates was used with TaqMan Gene Expression Assays, excellent precision across technical replicates was seen and highly reliable gene expression data were generated—comparable to that from purified total RNA.

Figure 9 (See Sidebar). Differential Expression of EGFR is Detected with Both Cells-to-cDNA™ II Lysate and with Purified Total RNA. Ct vs. Cell Number for an EGFR TaqMan® Gene Expression Assay with HeLa or Raji cell lysates or purified RNA show virtually identical results.

AM2222,AM8723

Applied Biosystems TaqMan® Gene Expression Assays

Designed for efficiency, specificity, and sensitivity using Applied Biosystems’ powerful bioinformatics pipeline and proprietary software algorithms, TaqMan Gene Expression Assays use TaqMan probes (5’ nuclease chemistry) that feature a nonfluorescent quencher and incorporate MGB (Minor Groove Binder) technology. There are over 781,000 pre-designed, pre-optimized assays to choose from for human, mouse, rat, Arabidopsis, Drosophila, C. elegans, Rhesus macaque, and C. familiaris (dog) genes. In addition, Custom TaqMan Gene Expression Assays are available. Each assay is a ready-to-use 20X mixture of PCR primers and probe designed for amplification using universal cycling conditions so that any combination of assays can be simultaneously run in a single thermal cycler. Just add TaqMan Universal PCR Master Mix (with or without AmpErase® UNG) and your cDNA sample to generate accurate, reproducible, and truly quantitative gene expression data.

Gene Expression Analysis of EGFR Directly From Cell Lysates

It has been well established that EGFR is highly expressed in many epithelial tumors and solid tumor-derived cell lines such as HeLa [1]. On the contrary, published flow cytometry evidence indicates that EGFR is not expressed in Raji cells derived from Burkitts lymphoma [2]. Furthermore, Moroni [3] found that EGFR is not expressed in EB-3 cells derived from a different B-cell lymphoma. Our experiments compared EGFR expression in HeLa and Raji cells and confirmed the published observations. We found significant differential expression of EGFR between HeLa and Raji cells; both Cells-to-cDNA™ II lysate and purified total RNA data show an approximately 10 cycle difference in Ct values (Figure 9).